Methods, compositions and apparatuses for facilitating regeneration

ABSTRACT

A polypeptide having at least 70% homology, in particular 80%, 90% or 95% homology to the polypeptide of SEQ ID No 2 representing the wild-type of the protein insect metalloproteinase inhibitor IMPIa and having at least one mutation at position  35, 36  and/or  39  of the amino acid sequence of IMPIa and the polypeptide having an IC 50  value to thermolysine of less than the IC 50  value of IMPIa wherein—the nonpolar amino acid isoleucine at position  35  of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of leucine, methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine, asparagine, glutamine, histidine, lysine and arginine; and/or—the nonpolar amino acid isoleucine at position  36  of IMPIa is replaced either by a nonpolar amino acid selected from the group consisting of valine, phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine, histidine, lysine and arginine; and/or—the polar amino acid position  39  of IMPIa is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional Application Ser. No. 61/055,846, METHODS, COMPOSITIONS AND APPARATUS FOR FACILITATING REGENERATION, filed on May 23, 2008, and is hereby incorporated in its entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not applicable.

FIELD OF THE INVENTION

The present invention relates in general to methods, compositions and apparatus for promoting tissue and organ regeneration, and in particular to preventing cells from inhibiting regenerative processes to obtain the recognized benefits in health and function associated with the results of regeneration.

BACKGROUND OF THE INVENTION

Tissue and organ regeneration research has focused on the need to stimulate regeneration by activating stem cells by soluble factors or treat partially- or non-functional cells to improve their function, e.g. by breaking damage-related crosslinks. Such research has overlooked the need to remove inhibitory effects.

Aging results from a combination of factors, but regeneration can overcome aging effects, if and to the extent that regenerative stem cells are functional. The stem cells, which replace cells, re-grow structures and renew the tissues of the body after normal wear-and-tear, give rise to replacement cells, and even structures, like hair follicles. In fact, if all stem cells in the body were destroyed, death would follow in a matter of days.

However, in a variety of diseases, malfunctions (such as male pattern baldness) and tissue injuries, cell types are not observed to be replaced when damaged or nonfunctional. Stem cell transplant therapy is proposed for such conditions, although it is not always successful.

The art has been left with the question as to why, then, does the body succumb to injury and aging when it has a mechanism for regeneration.

SUMMARY OF THE INVENTION

The present invention provides apparatus, compositions and methods for removing cells that interfere with regenerative processes by blocking locations in a tissue where progeny of stem cells can improve function. The present invention also reduces the proportion of partially- and non-functional cells without regard to location in a tissue.

Functionality according to the present invention is defined as the state of operation of a cell of the same type in a selected healthy individual.

The apparatuses, compositions and methods according to the present invention promote regenerative processes by differentially killing cells based inversely on the degree of functionality of the cells (i.e., the less functionality the more likely to be killed), and then applying that technique. This cell killing technique preferentially preserves proliferating cells. For example, any of lipofuscin, glycarion end-products or cell stiffness can be selected as a proliferation-preserving marker of partial- or non-functionality. Cell killing technologies directed against such markers can include, respectively, lasers/intense light, antibodies, and ultrasound. Cells can be killed according to the present invention by physical, electromagnetic, chemical or biological techniques, for example. Physical techniques include without limitation ultrasound and other oscillatory methods for disrupting cell membranes or structures leading to cell death. Electromagnetic techniques include without limitation and as targeted by sensitizers (such as absorbent nanoparticles, for example) EMF (see, e.g., Litovitz, U.S. Pat. No. 7,367,988 for EMF methods), high intensity light, radio waves microwaves, lasers, magnetism and ionizing radiation. Chemical techniques include without limitation toxic nanoparticles, chemical toxins and structure removal compounds such as β-aminopropionitrile. Biological techniques include without limitation antibodies against partially-functional or non-functional cells and variations and modifications thereof, such as toxin conjugates and natural killer cells modified to express target-specific antibodies. Techniques can be combined as determined to be effective (e.g. see McHale et al., U.S. Pat. No. 6,821,274 for sensitization to ultrasound by EMF treatment). Apparatuses, methods and compositions according to the present invention can be used sequentially or simultaneously in combination as monitoring determines to be effective for promoting regeneration.

Preferably, the apparatuses, compositions and methods selectively kill partially and/or non-functional cells versus functional cells of the same cell type to the extent that, upon removal of the killed cells by disintegration or scavenging, functional cells replace them. The cell killing apparatus, compositions and methods according to the present invention must preferentially preserve proliferating, functional cells and must be of a degree that avoids excessive inflammatory responses.

Evaluation of improvement or maintenance of a desired result can be used to direct the frequency of reapplication of the apparatus, compositions and methods according to the present invention. The application and reapplication can be determined with the goal of gradual improvement to avoid overwhelming natural mechanisms, such as removal of cells/debris by scavenging cells.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE illustrates apparatus according to the present invention.

DETAILED DESCRIPTION

Higher multicellular organisms behave as communities of specialized cells that work together not to preserve each individual cell, but to preserve the organism as a whole. In humans during development, cells that are “in the way” of development are removed by programmed cell death, apoptosis, to benefit tissues, organs or the organism as a whole. Even when an organism reaches maturity, certain damaged cells, such as damaged blood cells are destroyed by the body to make way for replacements. The replacement cells are derived from stem cells. Accordingly, in a mature organism, cell turnover is the key to maintenance of a youthful/functional whole.

This turnover can come at the expense of destruction of functional cells to the extent that the destruction does not degrade the function of the organism over time. Thus, such destruction must preferentially target non-proliferating cells that are partially- or non-functional, but it need not absolutely avoid killing functional and proliferating cells. The goal is to preserve the community of cells (e.g. organism) not individual cells. Proliferating cells include both cells that are dividing and cells, such as stem cells, that divide normally when stimulated to do so.

While stem cells participate in mundane tissue replacement, injury can also be an occasion for release of a factor or factors to stimulate stem cell proliferation and differentiation. Stem cells give rise to cells which heal the injury, for example, healing a cut in an epithelium.

However, stem cell division and differentiation would be abnormal, even tumorigenic, in the presence of a normal complement of cells. Accordingly, there are mechanisms to prevent excessive proliferation. For example, the presence of a cell at a location in an epithelium prevents replacement at that position. An example of this sort of phenomenon is contact inhibition where cells cease proliferating when they come in contact with other cells. The phenomenon can be generalized to a rule that, to facilitate stem cell proliferation and differentiation for regeneration of cells at a location, there can not be a cell or structure at the location in the tissue.

In some cases, the human body includes cells for tearing down a structure, such as osteoclasts in bone, as well as cells for building up a structure, such as osteoblasts in bone. It is the balance between the activities of the two types of cells that determines the extent of the resulting structure. To the extent that any intrinsic mechanisms do not remove cells/structure for periodic renewal, the present invention provides for removal to promote regeneration.

The cell at the location that inhibits stem cell action can be fully functional, partially functional, or non-functional. Dead cells can be removed by scavenging macrophages, thus allowing for replacement, but a malfunctioning cell may remain despite deleterious effects on the subject (i.e., the organism of which they are a part). A partially or non-functional cell, i.e. a malfunctioning cell, can not be apoptotic, and, thus, can not stimulate clearance by macrophages on its own. Such malfunctioning cells are killed according to the present invention for removal by the body's natural processes.

Therapeutic killing of cells in cancer therapy is targeted against proliferating cells, the exact opposite of the present invention. According to the present invention, action against cancer is provided by stimulating proliferation of stem cells so that error-correcting mechanisms that function during cell division can correct mutations that otherwise might accumulate in a non-dividing cell.

Without limitation, partially or non-functional cells according to the present invention can fail to be fully functional due to damage, such as free radical damage, or cross-linkage as a result of reaction with sugars, i.e. glycation. Cells that are partially or non-functional due to a genetic makeup that is shared by stem cells of a subject can be replaced by exogenous stem cells having a fully functional genetic makeup.

Blocking stem cell action by a cell or structure at a location in a tissue interferes with the action of endogenous and transplanted stem cells. A structure that blocks stem cell action need not be a cell. Non-cellular material, such as scar tissue, can block such regeneration. This can explain failures in regeneration and in stem cell transplantation. To the extent feasible under a given situation, non-cellular blocking structures can be removed according to the present invention.

With removal of blocking cellular and/or non-cellular materials, appropriate regenerative cells, such as stem cells, are retained or supplemented by transplantation in order to permit regeneration. “Fully functional” is defined as the degree of a specified function for a particular cell type exhibited by an available progeny of a stem cell in a subject with or without stem cell transplantation, whichever is greater.

An example of an apparatus according to the present invention is illustrated in FIG. 1. Effector 10 is a device for killing cells. Effector 10 can be, without limitation, ultrasound equipment or a device for antibody administration, such as a drip apparatus. Control 20 is a device for regulating the operation of effector 10 according to preset parameters and/or as modified to ensure safety or effectiveness. Without limitation, control 20 can be a control panel of effector 10. Monitor 30 provides information regarding the degree of inflammatory response and/or other important factors in the condition of the subject to which effector 10 is applied. Information from monitor 30 can be used to adjust control 20 and thereby to adjust or change the operation of effector 10. Monitor 30 can be, without limitation, a thermometer connected to control 20.

A technique according to the present invention is selected to preferentially kill partially functional or nonfunctional cells or to remove non-cellular compositions, as opposed to indiscriminate killing, which has as great an effect on functional cells. A technique may be selected according to the present invention by exposing functional and partially functional and/or non-functional cells to the technique and choosing concentrations, intensities and characteristics such as wavelength, frequency, wave shape, continuity and treatment duration. The technique to be applied to a particular subject can be chosen on the basis of identifying an acceptable selectivity for partially functional and/or non-functional cells versus functional cells.

Suitable selection methods and criteria are readily available to those skilled in the art. Such selection methods are routinely applied by those of skill in the relevant arts to select laser treatment levels for removing blemishes, treating cancers by radiation therapy, selecting monoclonal antibodies, selecting toxins to be used therapeutically, and selecting ultrasound properties for therapy, for example. According to the present invention, selected techniques discriminate functional versus non-functional and/or partially functional cells of the same cell type as the functional cells.

Techniques that act upon differences between functional and partially or non-functional cells can be based upon cellular properties associated with dysfunction, such as cross-linking, membrane stiffness and brown coloration associated with lipofuscin in aged or senescent cells as opposed to nascent, dividing or functional cells. Techniques such as ultrasound, targeted to harmonic frequencies of cross-linked cell membranes or components, can be used according to the present invention. Likewise, techniques such as lasers or intense light of a wavelength preferentially absorbed by partially functional or non-functional cells can be used according to the present invention.

Techniques according to the present invention can be used to localize therapy where needed. Localization can be accomplished by, without limitation, computer assisted tomography, magnetic resonance imaging, and positron emission tomography. Most preferably, techniques according to the present invention can be applied to the whole organism without the need for localization.

Once techniques are chosen for one or more targets, the techniques can be applied periodically, particularly at a low intensity or concentration, to maintain or increase a positive balance between functional versus partially or non-functional cells. Gradual versus precipitate cell killing can aid in avoiding toxic effects from high levels of cellular breakdown products and/or deleterious effects of an inflammatory response.

In addition to therapeutic applications, it is intended that non-therapeutic, non-human and industrial applications be included within the scope of the present invention. Cosmetic applications, diagnostic applications and veterinary applications are also contemplated. Repeatedly practicing the method according to the present invention at a low level can be coupled with monitoring to determine the degree of improvement as a diagnostic measure of the component of a condition due to damaged cells versus genetic factors. With respect to tissue and cell culture applications, for example, destruction of blocking cells can permit the resulting dead cells and debris to be washed away. In this way, productivity of cell and tissue cultures can be increased by increasing the relative proportion of productive cells versus non-productive cells.

EXAMPLES

The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art that would similarly permit one to successfully perform the intended invention.

Example 1

In an embodiment of the present invention glycation end-products, crosslinks created by sugars bonded to proteins, are selected as indicia of an accumulation of cellular damage correlated with partial- or non-functionality. Antibodies against such glycation end-products can be raised according to methods well known to those skilled in the art (e.g. Abed et al., U.S. Pat. No. 6,380,165; Bucala, U.S. Pat. No. 5,702,704) and humanized monoclonal antibodies retaining constant regions which permit destruction of targeted cells by the immune system can be produced for injection, also according to well known methods (e.g. Basi et al., U.S. Pat. No. 7,256,273). Antibodies can be screened for effectiveness according to the present invention by labeling them and applying them separately to untreated cells versus cells incubated with a sugar such as ribose used to induce formation of glycation end-products. Binding of the antibodies to a higher degree to the cells previously incubated with sugar as opposed to the cells not treated with sugar indicates preferential effect against the selected target.

Antibodies produced as described above can be administered to a subject intravenously with monitoring to determine that inflammatory responses such as fever or swelling do not exceed limits well known to be safe. This process can be repeated at intervals to maintain a level of regeneration. The process can be focused to remove partially- and/or non-functional cells a particular location (e.g. where stem cell transplantation is targeted).

Evaluation of improvement or maintenance of a desired result can be used to direct the frequency of reapplication of the antibodies according to the present invention. The application and reapplication can be determined with the goal of gradual improvement to avoid overwhelming natural mechanisms, such as removal of cells/debris by scavenging cells.

Example 2

In another embodiment of the present invention, glycation end-products, crosslinks created by sugars bonded to proteins, are selected as indicia of an accumulation of cellular damage correlated with partial- or non-functionality. This crosslinking manifests itself in a stiffening of the cells. Those in the art understand stiffness to distinguish types of proliferating versus non-proliferating cells (e.g. Kas et al., U.S. Pat. No. 6,067,859).

Ultrasound apparatus can be used according to practices well known to those skilled in the art to destroy cells by vibrational techniques (e.g. Chapelon et al., U.S. Pat. No. 5,601,526). Ultrasound parameters (e.g. frequency, power, and pulsation) can be screened for effectiveness in selectively destroying stiffer cells according to the present invention by application to untreated cells versus cells incubated with a sugar such as ribose used to induce formation of glycation end-products. Vibrational versus thermal destruction by ultrasound is preferred according to the present invention. Parameters selected for preferential destruction of sugar-treated cells as opposed to the cells not previously treated with sugar indicates preferential effect against the selected target.

Ultrasound as described above can be applied to a subject with monitoring to determine that inflammatory responses such as fever or swelling do not exceed limits well known to be safe. This process can be repeated at intervals to maintain a level of regeneration. The process can be focused to remove partially- and/or non-functional cells a particular location (e.g. where stem cell transplantation is targeted).

Evaluation of improvement or maintenance of a desired result can be used to direct the frequency of reapplication of ultrasound according to the present invention. The application and reapplication can be determined with the goal of gradual improvement to avoid overwhelming natural mechanisms, such as removal of cells/debris by scavenging cells.

While the present invention has been described in terms of preferred embodiments, it is not intended that the present invention be limited to the embodiments described herein, but, rather, that the present invention include all embodiments within the scope of die appended claims as properly construed. 

What is claimed is: 1-9. (canceled)
 10. A humanized antibody that binds to a glycation end-product of a cell, wherein the antibody is conjugated to a toxin.
 11. The antibody of claim 10, wherein the antibody is monoclonal.
 12. The antibody of claim 10, wherein the toxin is a toxic nanoparticle.
 13. The antibody of claim 10, wherein the toxin is a chemical toxin.
 14. The antibody of claim 10, wherein the toxin is p-aminopropionitrile.
 15. The antibody of claim 10, wherein the cell is a senescent cell.
 16. The antibody of claim 10, wherein the antibody is monoclonal, the toxin is a chemical toxin, and the cell is a senescent cell.
 17. A composition comprising a humanized antibody that binds to a glycation end-product of a cell, wherein the antibody is conjugated to a toxin.
 18. The composition of claim 17, wherein the antibody is monoclonal.
 19. The composition of claim 17, wherein the toxin is a toxic nanoparticle.
 20. The composition of claim 17, wherein the toxin is a chemical toxin.
 21. The composition of claim 17, wherein the toxin is p-aminopropionitrile.
 22. The composition of claim 17, wherein the cell is a senescent cell.
 23. The composition of claim 17, wherein the antibody is monoclonal, the toxin is a chemical toxin, and the cell is a senescent cell.
 24. A method of making an antibody-toxin conjugate, comprising: conjugating a toxin to a humanized antibody that binds to a glycation end-product of a cell.
 25. The method of claim 24, wherein the antibody is monoclonal.
 26. The method of claim 24, wherein the toxin is a toxic nanoparticle.
 27. The method of claim 24, wherein the toxin is a chemical toxin.
 28. The method of claim 24, wherein the cell is a senescent cell.
 29. The method of claim 24, wherein the antibody is monoclonal, the toxin is a chemical toxin, and the cell is a senescent cell. 